do not necessarily reflect the views of UKDiss.com.
Elucidating the Role of the ERM Family Adaptors, Ezrin, Radixin and Moesin, in Breast Cancer Cell Drug Resistance and Metastasis
- Therapeutic strategies for breast cancer treatment
- Overview of breast cancer
Breast cancer is the most common cancer among Canadian women, accounting for an average of 72 new diagnoses each day nationwide1. Breast cancer originates from cells in the breast that begin to grow uncontrollably and deviate from the normal process of cell division. The majority of breast cancers arise from cells that line the mammary ducts which carry milk from the mammary glands to the nipple. This type of breast cancer is called ductal carcinoma. In contrast, lobular carcinoma originates from the cells of the lobules, which are the milk-producing glands of the breast. Both of these breast cancers possess the ability to progress to an invasive metastatic stage in which treatment is often unsuccessful.
Staging is used to describe a cancer based upon the amount of cancerous tissue present and where in the body the cancer was first diagnosed. The most common staging system for breast cancer is the TNM system which consists of five stages. In situ breast cancer, which describes cancer cells that are non-invasive, is labelled as “stage 0” while metastatic breast cancer that has spread to other parts of the body, such as the bone, liver, lungs or brain, is labelled as “stage 4” and correlates to the most devastating prognosis.
As with many diseases, prognosis and survival for breast cancer patients depends on a wide variety of factors including age and menopausal status at diagnosis, medical history, stage of the cancer and the treatment strategies employed. Treatment options often revolve around the expression status of the estrogen receptor (ER), human epidermal growth factor type 2 receptor (HER2) and progesterone receptor (PR). When breast cancer has progressed to a metastatic stage, more aggressive treatment options such as combination chemotherapy are often required.
The era of modern chemotherapy can be traced directly to the discovery in the 1940s of nitrogen mustard as an effective cancer treatment2. A chemotherapeutic approach to treating cancer is based upon the concept that tumours, due to their tendency to display an increased rate of cell division, are more susceptible to certain toxins than normal tissues. These chemotherapeutics typically interfere with processes important in cell proliferation, particularly DNA replication, to trigger programed cell death pathways and eradicate cancer progression. This strategy was founded upon observations of autopsies of soldiers who were exposed to sulphur mustard gas during the First World War and displayed profound lymphoid hypoplasia and myelosuppression. Originally proposed by Louis Goodman and Alfred Gilman, a novel treatment strategy for a patient with advanced non-Hodgkin’s lymphoma proceeded by injecting into the bloodstream the closely related compound, nitrogen mustard3. Although only a few weeks of remission were observed before further disease progression, the ground-breaking principle that certain toxins could be administered systemically to promote tumour regression was established.
Following the end of the Second World War, an additional innovative approach to cancer drug therapy emerged as the effects of folic acid on patients with acute lymphoblastic leukaemia (ALL) were explored4. By blocking the function of folate-requiring enzymes, the folate analogue methotrexate became the first drug to successfully induce remission in children with ALL5. Methotrexate was subsequently proved to have anti-tumour activity in a wide variety of epithelial malignancies, including breast and ovarian cancers.
These discoveries quickly progressed and in the late 1960s evolved into more specific treatment strategies for metastatic breast cancer when combination chemotherapy in the adjuvant setting emerged at the forefront of cancer research6. In 1973, a clinical trial utilized combination chemotherapy with cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) to treat women with operable breast cancer7. Preliminary findings showed that patients within the adjuvant CMF group displayed significantly better overall survival than those who received only a mastectomy7. It was found that administering a combination of drugs, rather than a single agent, was a more effective treatment strategy against both metastatic cancer and for patients at high risk of relapse after primary surgical treatment4.
These foundational discoveries provided the basis of modern systemic chemotherapy, which are currently administered for treatment of a plethora of different cancers. Today, anthracyclines such as doxorubicin, are commonly used as chemotherapeutic agents to treat breast cancer. This drug prevents the DNA double helix from being relaxed out of its supercoiled formation by blocking the activity of the enzyme topoisomerase II. This mechanism arrests the cell replication process in tumour cells.
- Additional therapeutic approaches
Recent discoveries within the field of medical oncology have provided valuable insights into the heterogeneity of breast tumours, key oncogenic drivers and the role of the immune system in breast cancer8. These developments have allowed many novel therapeutic strategies to emerge. As immune escape is a well-known hallmark of cancer, researchers have developed immunotherapy treatments that work to reactivate the host immune system to attack and eradicate tumours9. The immune system normally uses regular “checkpoints” to inhibit unnecessary T-cell activation in order to prevent autoimmunity, however this system becomes dysregulated in cancer. As a result, drugs that target these “checkpoints” hold a lot of potential for cancer treatments. The cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death protein (PD-1) are members of the same class of co-inhibitor receptors expressed on activated T cells. As negative regulators of T-cell function, these proteins works to attenuate further T-cell activation during antigen presentation, as well as regulate immune reactions downstream within the tumour microenviornment8. Antagonistic monoclonal antibodies have been developed to block both CTLA-4 and PD-1 activation, proving to be promising therapeutics for breast cancer, especially when used in combination8.
Inhibition of enzymes by means of small molecules, such as tyrosine kinase inhibitors (TKIs), has also been important in recent therapeutic developments. TKIs work to inhibit the initial autophosphorylation of the kinase domain upon ligand binding, effectively inhibiting downstream signals for survival and proliferation pathways10. Lapatinib is the first TKI developed to treat breast cancer that targets both HER2, a surface protein critical to cell signaling pathways and the epidermal growth factor receptor (EGFR)11.
- Overview of ERM proteins
- Structure of ERM proteins
The ezrin-radixin-moesin (ERM) family is a class of highly homologous proteins involved in linking the plasma membrane to the cortical actin cytoskeleton. Through evolution, this family has been greatly conserved, presenting more than 75% amino acid identity that are shared between ezrin, radixin and moesin12. Figure 1 illustrates that structurally, ERMs are characterized by the presence of an approximately 300 amino acid long plasma membrane-associated Four point one, ERM (FERM) domain at the amino terminus, also known as the N-terminal ERM association domain (N-ERMAD)13,14. Revealed through X-ray crystallography, the FERM domain consists of F1, F2 and F3 subdomains which fold to form a cloverleaf structure15. This FERM domain is directly followed by a central
2. Goodman, L. S. et al. Nitrogen mustard therapy. J. Am. Med. Assoc. 132, 126 (1946).
3. Gilman, A. The initial clinical trial of nitrogen mustard. Am. J. Surg. 105, 574–578 (1963).
4. Chabner, B. A. & Roberts, T. G. Chemotherapy and the war on cancer. Nature Reviews Cancer 5, 65–72 (2005).
5. Farber, S., Diamond, L. K., Mercer, R. D., Sylvester, R. F. & Wolff, J. A. Temporary Remissions in Acute Leukemia in Children Produced by Folic Acid Antagonist, 4-Aminopteroyl-Glutamic Acid (Aminopterin). N. Engl. J. Med. 238, 787–793 (1948).
6. DeVita, V. T. & Chu, E. A history of cancer chemotherapy. Cancer Research 68, 8643–8653 (2008).
7. Rossi, A., Bonadonna, G., Valagussa, P. & Veronesi, U. Multimodal treatment in operable breast cancer: Five-year results of the CMF programme. Br. Med. J. (Clin. Res. Ed). 282, 1427–1431 (1981).
8. Cox, K., Alford, B. & Soliman, H. Emerging Therapeutic Strategies in Breast Cancer. South. Med. J. 110, 632–637 (2017).
9. Hanahan, Douglas, Weinberg, R. Hallmarks of Cancer. Cell 100, 57–70 (2000).
10. Hynes, N. E. & Lane, H. A. ERBB receptors and cancer: The complexity of targeted inhibitors. Nature Reviews Cancer 5, 341–354 (2005).
11. Johnston, S. R. D. & Leary, A. Lapatinib: A novel EGFR/HER2 tyrosine kinase inhibitor for cancer. Drugs of Today 42, 441–453 (2006).
12. Gary, R., Bretscher, A. & Gary…, R. Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol Biol Cell 6, 1061–1075 (1995).
13. Ponuwei, G. A. A glimpse of the ERM proteins. J. Biomed. Sci. 23, (2016).
14. Fehon, R. G., McClatchey, A. I. & Bretscher, A. Organizing the cell cortex: The role of ERM proteins. Nature Reviews Molecular Cell Biology 11, 276–287 (2010).
15. Bretscher, A., Edwards, K. & Fehon, R. G. ERM proteins and merlin: Integrators at the cell cortex. Nature Reviews Molecular Cell Biology 3, 586–599 (2002).
16. Pore, D. & Gupta, N. The ezrin-radixin-moesin family of proteins in the regulation of B-cell immune response. Crit. Rev. Immunol. 3, 973–982 (2016).
17. Hamada, K. et al. Structural basis of adhesion-molecule recognition by ERM proteins revealed by the crystal structure of the radixin-ICAM-2 complex. EMBO J. 22, 502–514 (2003).
18. Pearson, M. A., Reczek, D., Bretscher, A. & Karplus, P. A. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell 101, 259–270 (2000).
19. Trofatter, J. A. et al. A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor. Cell 72, 791–800 (1993).
20. Rouleau, G. A. et al. Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2. Nature 363, 515–521 (1993).
21. Cooper, J. & Giancotti, F. G. Molecular insights into NF2/Merlin tumor suppressor function. FEBS Letters 588, 2743–2752 (2014).
22. Nakamura, N. et al. Phosphorylation of ERM proteins at filopodia induced by Cdc42. Genes to Cells 5, 571–581 (2000).
23. Belkina, N. V., Liu, Y., Hao, J.-J., Karasuyama, H. & Shaw, S. LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation. Proc. Natl. Acad. Sci. 106, 4707–4712 (2009).
24. Matsui, T. et al. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J. Cell Biol. 140, 647–657 (1998).
25. Ng, T. et al. Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility. EMBO J. 20, 2723–2741 (2001).
26. Bretscher, A., Chambers, D., Nguyen, R. & Reczek, D. ERM-MERLIN AND EBP50 PROTEIN FAMILIES IN PLASMA MEMBRANE ORGANIZATION. Annu. Rev. Cell Dev. Biol. 113–143 (2000). doi:10.1146/annurev.cellbio.16.1.113
27. Speck, O., Hughes, S. C., Noren, N. K., Kulikauskas, R. M. & Fehon, R. G. Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity. Nature 421, 83–87 (2003).
28. Krieg, J. & Hunter, T. Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin. J. Biol. Chem. 267, 19258–19265 (1992).
29. Wakayama, T., Nakata, H., Kurobo, M., Sai, Y. & Iseki, S. Expression, localization, and binding activity of the ezrin/radixin/moesin proteins in the mouse testis. J. Histochem. Cytochem. 57, 351–362 (2009).
30. Yonemura, S. et al. Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J. Cell Biol. 140, 885–895 (1998).
31. Legg, J. W., Lewis, C. A., Parsons, M., Ng, T. & Isacke, C. M. A novel PKC-regulated mechanism controls CD44-ezrin association and directional cell motility. Nat. Cell Biol. 4, 399–407 (2002).
32. Helander, T. S. et al. ICAM-2 redistributed by ezrin as a target for killer cells. Nature 382, 265–268 (1996).
33. Weinman, E. J., Hall, R. A., Friedman, P. A., Liu-Chen, L.-Y. & Shenolikar, S. THE ASSOCIATION OF NHERF ADAPTOR PROTEINS WITH G PROTEIN–COUPLED RECEPTORS AND RECEPTOR TYROSINE KINASES. Annu. Rev. Physiol. 68, 491–505 (2006).
34. Chirivino, D. et al. The ERM proteins interact with the HOPS complex to regulate the maturation of endosomes. Mol. Biol. Cell 22, 375–385 (2011).
35. Dransfield, D. T. et al. Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO J. 16, 35–43 (1997).
36. Mackay, D. J. G., Esch, F., Furthmayr, H. & Hall, A. Rho- and Rac-dependent assembly of focal adhesion complexes and actin filaments in permeabilized fibroblasts: An essential role for ezrin/radixin/moesin proteins. J. Cell Biol. 138, 927–938 (1997).
37. Hatzoglou, A. et al. Gem associates with Ezrin and acts via the Rho-GAP protein Gmip to down-regulate the Rho pathway. Mol.Biol.Cell 18, 1242–1252 (2007).
38. Reczek, D. & Bretscher, A. Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP. J. Cell Biol. 153, 191–205 (2001).
39. Hamada, K. et al. Crystallization and preliminary crystallographic studies of RhoGDI in complex with the radixin FERM domain. Acta Crystallogr. Sect. D Biol. Crystallogr. 57, 889–890 (2001).
40. Schwartz, M. Rho signalling at a glance. J. Cell Sci. 117, 5457–5458 (2004).
41. Kunda, P., Pelling, A. E., Liu, T. & Baum, B. Moesin Controls Cortical Rigidity, Cell Rounding, and Spindle Morphogenesis during Mitosis. Curr. Biol. 18, 91–101 (2008).
42. Carreno, S. et al. Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells. J. Cell Biol. 180, 739–746 (2008).
43. Pilot, F., Philippe, J. M., Lemmers, C. & Lecuit, T. Spatial control of actin organization at adherens junctions by the synaptotagmin-like protein Btsz. Nature 442, 580–584 (2006).
44. Molnar, C. & de Celis, J. F. Independent roles of Drosophila Moesin in imaginal disc morphogenesis and hedgehog signalling. Mech. Dev. 123, 337–351 (2006).
45. Serano, J. & Rubin, G. M. The Drosophila synaptotagmin-like protein bitesize is required for growth and has mRNA localization sequences within its open reading frame. Proc. Natl. Acad. Sci. USA 100, 13368–13373 (2003).
46. Christofori, G. New signals from the invasive front. Nature 441, 444–450 (2006).
47. Hunter, K. W. Ezrin, a key component in tumor metastasis. Trends in Molecular Medicine 10, 201–204 (2004).
48. Ren, L. et al. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC. Oncogene 28, 792–802 (2009).
49. Jeanes, A., Gottardi, C. J. & Yap, A. S. Cadherins and cancer: How does cadherin dysfunction promote tumor progression? Oncogene 27, 6920–6929 (2008).
50. Yu, Y. et al. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat. Med. 10, 175–181 (2004).
51. Elliott, B. E., Meens, J. A., SenGupta, S. K., Louvard, D. & Arpin, M. The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells. Breast Cancer Res. 7, (2005).
52. Khanna, C. et al. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat. Med. 10, 182–186 (2004).
53. Sarrió, D. et al. Abnormal ezrin localization is associated with clinicopathological features in invasive breast carcinomas. Breast Cancer Res. Treat. 98, 71–79 (2006).
54. Elzagheid, A. et al. Intense cytoplasmic ezrin immunoreactivity predicts poor survival in colorectal cancer. Hum. Pathol. 39, 1737–1743 (2008).
55. Köbel, M. et al. Ezrin expression is related to poor prognosis in FIGO stage I endometrioid carcinomas. Mod. Pathol. 19, 581–587 (2006).
56. Naba, A., Reverdy, C., Louvard, D. & Arpin, M. Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering. EMBO J. 27, 38–50 (2008).
57. Crepaldi, T., Gautreau, A., Comoglio, P. M., Louvard, D. & Arpin, M. Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells. J. Cell Biol. 138, 423–434 (1997).
58. Arpin, M., Chirivino, D., Naba, A. & Zwaenepoel, I. Emerging role for ERM proteins in cell adhesion and migration. Cell Adhesion and Migration 5, 199–206 (2011).
59. Bartholow, T. L., Becich, M. J., Chandran, U. R. & Parwani, A. V. Immunohistochemical analysis of ezrin-radixin-moesin-binding phosphoprotein 50 in prostatic adenocarcinoma. BMC Urol. 11, (2011).
60. Chen, S.-D. et al. Knockdown of Radixin by RNA interference Suppresses the Growth of Human Pancreatic Cancer Cells in Vitro and in Vivo. Asian Pacific J. Cancer Prev. 13, 753–759 (2012).
61. Kahsai, A. W., Zhu, S. & Fenteany, G. G protein-coupled receptor kinase 2 activates radixin, regulating membrane protrusion and motility in epithelial cells. Biochim. Biophys. Acta – Mol. Cell Res. 1803, 300–310 (2010).
62. Haynes, J., Srivastava, J., Madson, N., Wittmann, T. & Barber, D. L. Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression. Mol. Biol. Cell 22, 4750–4764 (2011).
63. Kobayashi, H. et al. Clinical Significance of Cellular Distribution of Moesin in Patients with Oral Squamous Cell Carcinoma. Clin. Cancer Res. 10, 572–580 (2004).
64. Belbin, T. J. et al. Molecular profiling of tumor progression in head and neck cancer. Arch. Otolaryngol. – Head Neck Surg. 131, 10–18 (2005).
65. Zhu, X. et al. Moesin is a glioma progression marker that induces proliferation and Wnt/β-Catenin pathway activation via interaction with CD44. Cancer Res. 73, 1142–1155 (2013).
66. Estecha, A. et al. Moesin orchestrates cortical polarity of melanoma tumour cells to initiate 3D invasion. J. Cell Sci. 122, 3492–3501 (2009).
67. Canadian Cancer Statistics Advisory Committee. Canadian Cancer Statistics 2018 Special report on cancer incidence by stage. Toronto, ON: Canadian Cancer Society (2018). doi:10.1080/0141861021000039455
68. Coley, H. M. Mechanisms and strategies to overcome chemotherapy resistance in metastatic breast cancer. Cancer Treatment Reviews 34, 378–390 (2008).
69. Liu, Q., Xu, B. & Zhou, W. Correlation between chemotherapy resistance in osteosarcoma patients and PAK5 and Ezrin gene expression. Oncol. Lett. 15, 871–878 (2018).
70. Brambilla, D. et al. P-glycoprotein binds to ezrin at amino acid residues 149-242 in the FERM domain and plays a key role in the multidrug resistance of human osteosarcoma. Int. J. Cancer 130, 2824–2834 (2012).
71. PubMed. PubMed – NCBI. PubMed (2015). Available at: http://www.ncbi.nlm.nih.gov/books/NBK3827/#pubmedhelp.PubMed_Quick_Start.
72. Shalem, O., Sanjana, E. N., Hartenian, E. & Zhang, F. Genome-Scale CRISPR-Cas9 Knockout. Science (80-. ). 343, 84–88 (2014).
73. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nature Methods 11, 783–784 (2014).
74. LentiCRISPRv2 and lentiGuide-Puro: lentiviral CRISPR/Cas9 and single guide RNA. doi:10.1126/science.1247005
75. Toth, M., Sohail, A. & Fridman, R. Assessment of gelatinases (MMP-2 and MMP-9) by gelatin zymography. Methods Mol. Biol. 878, 121–135 (2012).